Preparation Monoclonal Anti-idiotype Antibody Of Zearalenone And Development Of Non-toxic ELISA Quantitative Detecting Technique

Objective:In order to develop a non-toxic ELISA quantitative detectingtechnique of zearalenone (ZEN) from the corn, a monoclonal anti-idiotype antibodyagainst zearalenone was prepared,and was used to replace toxin standardimmunoassays.Methods:1. The hapten ZEN-CMO derive from the action betweenzearalenone and carboxymathyloxime, and then identified using liquidchromatography/mass spectrometry. The hapten was coupled with carrier proteinovabumin and bovine serum albumin (BSA) to prepare artificial antigen by activeester method. Following identification by SDS-PAGE and UV scanning spectrum.The monoclonal antibodies against zearalenone were produced through immunizationand general hybridoma technology. And the titer and subtype of the antibodies wereidentified by indirect ELISA, and the sensitivity was identified by indirectcompetetive ELISA.2. The anti-ZEN mAb coupled with KLH was used as immunogen to immunizeBalb/c mouse, Fab' fragment from anti-ZEN mAb that was prepared by immobilizedpepsin cleavage,and was used as coating antigen when the serum,supernatants andascites were detected by indirect ELISA The monoclonal anti-idiotype antibodiesagainst zearalenone were produced through general hybridoma technology. And thetiter of the antibodies were identified by indirect ELISA, and the sensitivity wasidentified by indirect competetive ELISA.3. Reseach the correlation between ZEN anti-idiotype antibody and ZEN toxin,and develop a non-toxic ELISA quantitative detecting technique of zearalenone (ZEN)from the corn sample based on the idiotype and anti-idiotype reaction.Results:1.Preparation the artificial antigen ZEN-BSA and ZEN-OVA ofzearalenone toxin,it was succeeded that the antigen were coupled, identifying bySDS-PAGE and Uv Spectroscopy Scanning, the conjugation ratios were3.1:1and16.9:1,respectively. 2. ZEN-OVA was used as immunogen to immunize BALB/c mouse, and onehybridoma strain (named1G4) which could specifically secrete antibody of ZEN wasobtained by using cell fusion technique. The indirect ELISA titers of the cellsupernatant and ascites were1:6.4×10³and1:1.6×10⁵, respectively, the antibodiessubtypes was IgG2b, and the IC₅₀ to ZEN was10.2ng/ml,the detection limit was0.58ng/ml;3.It successfully obtained the Fab fragment of anti-ZEN mAb, and the Westernblot result showed that the MW of Fab fragment was50kD, also it maintain superiorbiological activity identified by indirect ELISA;4. The BALB/c mice were immunized with1G4-KLH conjugate, and sixhybridoma strains which could specifically secrete anti-idiotype antibodies of ZENwere obtained by using cell fusion technique. The indirect ELISA titers of the cellsupernatant and ascites were1:120and1:1.2×10⁵～1:2.0×10⁵, respectively. Theindirect ELISA results showed that the six antibodies competitive with ZEN binded toanti-ZEN Mab, thus they were β anti-idiotype antibody, and the IC₅₀ to ZEN was10.09～54.48ng/mL;5.Development the non-toxic ELISA quantitative detecting technique ofzearalenone applying monoclonal antibody and monoclonal anti-idiotype antibodyagainst ZEN,the replacement linear regress equation between monoclonalanti-idiotype antibody and ZEN standard: y=57.21x-7.7118, R2=0.990(y:concentrate of ZEN ng/mL x:concentrate of monoclonal anti-idiotype antibodyμg/mL).The lowest determined limit is2.63ng/mL,the linear range of ZEN was2.63～100.64ng/mL.The recovery of ZEN in corn samples test by non-toxic ZENELISA method established is between82.13%～88.41%,the CV of within group is8.25%～9.35%,the CV between groups is between6.54%～9.41%.Conclusion:1.The anti-ZEN McAb was successfully prepared through cellfusion technology, the characteristics of this McAb is with high antibody titer, highsensitivity, strong specificity；2.The monoclonal anti-idiotype antibodies against ZEN was successfully performed,and were identified as Ab2β;3.There is a strong correlation between monoclonal anti-idiotype antibodyagainst ZEN and ZEN standard, and was used to replace toxin standardimmunoassays;4. The non-toxic ELISA quantitative detecting technique of zearalenone wasdeveloped by applying ZEN monoclonal antibody and monoclonal anti-idiotypeantibody.